Process for typing HLA-B using specific primers and probes sets

ABSTRACT

The invention relates to a method for typing or subtyping one or more HLA-B alleles characterized by the sequence GCCA at position 30 to 33 of exon 2 (with said numbering being according to Zemmour and Parham, 1992), liable to be present in a sample, with said method comprising at least the following steps: (i) amplifying HLA-B alleles with at least one 5&#39; end amplification primer selected from the following list: 5&#39; -AGGTATTTCTACCCGCCA-3&#39; (B25P) or sequence variants thereof, in combination with an appropriate 3&#39; end primer being chosen from the same alleles as the above defined 5&#39; end primers, with said 5&#39; and 3&#39; end primers being possibly labelled; and, (ii) hybridizing the amplified product, being labelled during or after amplification, at appropriate conditions with one or more suitable probes selected from region 15 to 261 of the HLA-B exon 2 region, with said numbering being according to Zemmour and Parham 1992, (iii) washing at appropriate washing conditions, (iv) detecting the hybrids formed; and, (v) inferring the allele present from the observed hybridization pattern.

The invention relates to a process and reagents for DNA typing of HLA-Balleles.

The technical problem underlying the present invention is to provide aDNA typing method using specific primer and probe sets enabling thediscrimination of HLA-B alleles, especially those which are difficult todiscriminate by serological means.

The Human Leukocyte Antigen (HLA) system comprises a series of linkedgenes on the short arm of chromosome 6. Three classes of genes aredefined: class I antigens (HLA A, B, C) composed of an α chain noncovalently associated with β2 microglobulin, encoded on chromosome 15;class II antigens (DP, DQ, DR) composed of an α and a β chain; class IIIproducts which correspond to components of the complement system. ClassI and class II antigens are polymorphic transmembrane glycoproteins andshare a common immunologic role in antigen presentation. HLA class Irestricted presentation of foreign antigens leads to cytotoxic T cellreceptors in mature T lymphocytes. In addition, class I and class IIantigens play a crucial role in transplantation immunology and in thesusceptibility to autoimmune diseases.

Extensive polymorphisms exist at most loci. In view of the biologicaland medical importance of these antigens a highly sensitive and rapidtechnique for HLA typing is required. Different protocols have been useduntil now: serologic, cellular and DNA based restriction fragmentpolymorphism (RFLP) and recently also sequence specific oligonucleotide(SSO) hybridization methods. DNA typing by oligonucleotide hybridizationprovides the best direct definition of HLA polymorphisms next tocomplete sequence analysis. However, sequence analysis is expensive andtime consuming and hence not the method of choice for routineapplications.

Polymorphisms are of fundamental significance for the function of HLAantigens and will be mostly localized in exons coding for thefunctionally important extracellular domains.

For class I genes most of the polymorphisms are localized in theaminoterminal α1 and α2 domains. The α3 domain is a highly conservedimmunoglobin-like domain. A total of 40 HLA A, 64 HLA B, and 24 HLA Calleles have been identified (Zemmour and Parham, Tissue Antigens, 40:221-228,1992). Diversity between different alleles occurs in specificregions of the α1 and α2 domain. A patchwork pattern with shortstretches of homology between different alleles occurs. Geneticmechanisms such as homologous recombination and exon shuffling have leadto locus specific allelic diversity (Parham et al. PNAS (USA)85:4005-4009, 1988).

Different typing methods have been developed to discriminate between thedifferent alleles of the very polymorphic class I and class II loci. Anoverview of these different typing methods is given below:

Serology: In a microtoxicity test antisera to different HLA class I orclass II antigens are incubated with lysed purified lymphocytes. Lysedcells will be stained with eosin or other dyes while not-lysed cellswill remain unstained. This method is used for class I A, B, C allelesand class II DR and DQ alleles. DP alleles cannot be typed due to toolow level of expression and a limited availability of antisera. Thereaction for class II alleles is performed on purified B lymphocytes. Alimited determination of supertypic groups of alleles is possiblewithout further subtyping. Three alleles of the HLA C locus remainserologically undefined. Since epitopes on the HLA molecule aredetected, discrimination between the α and the β chains is impossibleand the αβ hetrodimer is identified. Allosera against class II moleculesare often anti-class I contaminated and need to be absorbed before use.Crossreactions between alleles on the same or on a different locusoccur, which makes analysis of the result difficult. Even if monoclonalantibodies are used the problem of crossreactions is not solved.Although this is a very rapid method (3 hrs for complete typing),incomplete and erroneous results are the main problems.

Cellular methods: a mixed lymphocyte reaction (MLR) has been developedbased on the proliferative response of T-cell cultures to stimulation byirradiated homozygous typing cells. Proliferation is measured byincorporation of H³ -Thymidine. This method is used for HLA class IItyping of DR and DQ alleles. DP typing is also impossible because of thelow level of its membrane expression. These analyses define the DWspecificities that further subdivide the serological specificities. HLADW specificities are determined by DR and DQ antigens but are almostalways associated with a particular allele on the DR and DQ locus. Asecondary MLR can be performed for HLA DP typing. This analysis is basedon in vitro secondary or memory responses. When lymphocytes haveresponded to irradiated stimular cells, after 10 days of culture theyrevert from blast cells expansion to the production of smalllymphocytes. These cells have the capacity to give a stronger andaccelerated response in culture to irradiated stimulator cells whichhave to be typed and which share the antigen with the first stimulatorcells that gave the initial positive reaction (Festenstein and Ollier,1987). Although very complete and correct, this analysis is very timeconsuming and difficult to perform.

Different DNA typing methods were developed that have the advantage notto be linked to surface expression of the antigens. An overview of theseDNA typing methods is given:

Restriction fragment length polymorphism (RFLP) methods: High molecularweight DNA is digested with several restriction enzymes, separatedaccording to size by gel electrophoresis, blotted to filters andhybridised to HLA DQA, DQB, DPB or DRB cDNA probes. A distinct patternof bands for the different alleles is obtained. Sequence analysis wasused to find the different restriction site polymorphisms and todetermine the different enzymes to be used. This method however has somedisadvantages: large amounts of high-molecular weight DNA are needed,not many alleles can be distinguished, use of several restrictionenzymes, detection of phenotypically irrelevant specific amino aciddifference.

Polymerase Chain Reaction (PCR) methods: since the sequences of allclass II alleles are known, locus-specific primers can be designed toamplify the polymorphic regions. After amplification, large amounts ofspecific sequences are analyzed by RFLP analysis or by SSOhybridization. The PCR product can be digested by different restrictionenzymes and the fragments separated on gel by electrophoresis. Analternative is the hybridization method. Sequence-specificoligonucleotides (SSO's) are designed. Hybridization can be performed ina conventional dot blot procedure. PCR products are covalently bound toa membrane and hybridised to ³² P labelled SSOs. Other labelling methodsare possible. Detection of positive signals is done by autoradiography.Since all. SSOs may differ in length and GC content differenthybridisation temperatures are maybe needed for the different SSOs inthe conventional dot-blot approach.

The conventional serological and cytological typing techniques for HLAclass I antigens are well-established. However, erroneous results mayoccur due to the specificity of the antisera, the presence ofauto-antibodies or medication. For HLA-B, typing problems arepredominantly experienced with the following types:

    ______________________________________    Serological Designation                        Corresponding allele    ______________________________________    B54 (22)            B*5401    B52 (5)             B*5201/B*52012    B78                 B*7801    B62 (15)            B*1501/B*1504    B75 (15)            B*1502    B72 (70)            B*1503    B71 (70)            not yet known    B46                 B*4601    B79                 B*7901    B58 (17)            B*5801    B53                 B*5301    B5102 (B5/B35)      B*5102    B5103 (BTA)         B*5103    ______________________________________

A DNA based typing system will greatly improve and accelerateidentification of these difficult HLA-B types. As a consequence, thiswill have a beneficial impact on the success-rate of organ andbone-marrow transplantations and the total costs involved. There isample evidence that a high correlation exists between the success-rateof transplantation and the HLA-B compatibility of the donor and therecipient.

In addition a more accurate HLA-B typing will also considerably improveor facilitate disease susceptibility studies and forensicinvestigations.

It should be noted that, in general, DNA typing methods should bepreferred over serological typing provided that an easy, rapid andreliable DNA typing method is available. This is due to the fact thatsome differences at the subtype level (which are usually detectable byDNA methods) might go undetected by current serological typing methods,although these differences might provoke allograft rejection(Fleischhauer et al., New Eng. J. Med. 323: 1818-1822, 1990).

In contrast with successful application of molecular biology for thedefinition of HLA class II genes, development of class I moleculartyping remains difficult. This is due to marked polymorphism, highcomplexity of nucleotides substitutions and presence of numerous nonclassical class I genes and pseudogenes existing in this region. Class Iantigens are characterised by cross-reactivity among different allelesmostly within HLA-A and -B antigens defining serologicalcross-reactivity groups (CREGs). The HLA-A and B alleles are dividedinto respectively five and ten classical CREG families. Up to now, allknown HLA-A specificities have been sequenced resulting in thedefinition of sequence homology explaining these cross-reactions and therecent capacity to use the polymerase chain reaction (PCR) usingsequence specific primers (SSP). In contrast, the HLA-B alleles arecharacterized by a greater polymorphism and by a higher level ofcross-reactivity. In addition, a substantial proportion of the B alleleshave not yet been sequenced. Correlation between cross-reactivity andDNA sequences present some discrepancies.

The number of publications dealing with PCR and DNA probe typing ofclass I alleles are limited as compared to those for class II. Exceptfor the paper of Yoshida et al. (1992, vide infra), the alleles whichare the subject of the present invention are not covered in thosestudies. Summers et al. (Hum. Immunol. 32: 176-182, 1991) described theuse of PCR of class I alleles for sequencing purposes. The combinationof PCR and classical dot hybridizations with oligonucleotide probes forthe discrimination of B44 alleles (B*4401, B*4402) was described byFleischhauer et al. (N. Eng. J. Med. 323: 1818-1822, 1990). Two researchgroups reported on the DNA typing and subtyping of HLA-B27 alleles (Hillet al., The Lancet, 337: 640-642, 1991; Dominguez et al., Immunogenetics36: 277-282, 1992). Hernandez-Vina et al. (Hum. Immunol. 33: 163-173,1992) described oligonucleotide typing subsequent to PCR amplificationfor HLA-A2 and HLA-A28 alleles; and a more general typing approach forHLA-A alleles using the amplification refractory amplification systemwas recently described by Krausa et al. (The Lancet, 341: 121-122,1993).

Yoshida et al. (Hum. Immunol. 34: 257-266, 1992) also combined PCR witha classical dot blot approach and/or single strand confirmationpolymorphism analysis. They designed PCR-primer and probe combinationsfor typing of 26 HLA-B specificities. Their typing approach is primarilybased on the use of differential amplification with primersdiscriminating between the Bw4 and Bw6 supratypes and the use of a HLA-Bspecific 5'-sided primer. With the primer sets and probes described,these authors did not discriminate the following alleles: B*5401, B*7801and B*7901, which are also difficult to type by conventional methods.Neither can they discriminate between B*5201 and B*52012 nor between B53and B51 alleles since sequence differences between these alleles arelocated outside the region amplified with their primers.

The present invention thus aims at providing a method for DNA typing orsubtyping one or more HLA-B alleles by a hybridization approach.

More particularly the present invention aims at a method for DNA typingand/or subtyping of those HLA-B alleles of which the correspondingserological-typing procedure poses problems or is impossible. The mostpredominant alleles for which the serological typing procedure posesproblems are the following:

    ______________________________________    Serological Designation                        Corresponding allele    ______________________________________    B54 (22)            B*5401    B52 (5)             B*5201/B*52012    B78                 B*7801    B62 (15)            B*1501/B*1504    B75 (15)            B*1502    B72 (70)            B*1503    B71 (70)            not yet known    B46                 B*4601    B79                 B*7901    B58 (17)            B*5801    B53                 B*5301    B5102 (B5/B35)      B*5102    B5103 (BTA)         B*5103    ______________________________________

According to another embodiment, the present invention aims at providingsequence specific oligonucleotides which allow the DNA typing of HLA-Balleles for which the serological typing procedure poses problems suchas selected from the above-presented list.

According to yet another embodiment, the present invention aims atproviding sequence specific primers which allow the DNA typing of HLA-Balleles for which the serological typing procedure poses problems suchas selected from the above-presented list.

The present invention also aims at providing compositions or solidsupports comprising at least one of the above-mentioned sequencespecific oligonucleotides and/or specific primers.

More particularly, the method of the invention aims at amplifying exon 2of a specific subset of HLA-B alleles, corresponding to the alleles forwhich the serological typing procedure causes problems, by means ofspecific primers (SPs) and subsequently hybridizing the amplifiedproducts to an appropriate set of sequence specific oligonucleotides(SSOs) covering the amplified region of the HLA-B exon 2.

The present invention also aims at providing kits for DNA typing ofHLA-B alleles for which the serological typing procedure poses problemssuch as selected from the above-presented list.

The present invention meets the above-mentioned aims by providing novelprocesses and reagents. More particularly, the present invention meetsthe above-mentioned needs by providing a specific set of specificprimers (SPs) and sequence specific oligonucleotides (SSOs), and kitsfor practicicing said methods, that toghether provide a rapid, simpleand precise system for typing the alleles at the HLA-B genes.

The novel process and reagents according to the invention may in turnlead to the discovery of previously unknown HLA-B alleles, which canalso be typed and identified by the present method. Also some othertypes (not listed in the table above) may cause typing difficulties byserology such as B76, B77, B61, B67 and B59. Although the nucleic acidsequences are not yet known, it is sometimes possible to predict whetheror not these specificities can be typed using the approach of thisinvention. This will be examplified further herein for B71.

The expression "typing or subtyping" is to be understood as determiningand/or discriminating the type or subtype present in a biologicalsample. By type or subtype is understood all variants which may bediscriminated by said typing method. In the case of discriminating thetype of one allele, the typing method determines the presence of saidspecific allele.

The officially recognized serological types (also called HLAspecificities) and their corresponding alleles (if the sequence isknown) are compiled in an annually updated reference list (Bodner etal., Tissue Antigens, 39:161-173, 1992).

The method of the invention is based on an amplification of exon 2 of asubset of HLA-B alleles possibly present in the sample with particularsets of amplification primers (also referred to as SPs). Subsequentlythe amplified products are hybridized to an appropiate set of DNA probes(also referred to as SSOs) after which the hybrids formed are detectedand the HLA-B type deduced from the hybridization pattern generated. Inthis approach, particularly aiming at the identification of types whichare difficult to distinguish or not distinguishable at all byserological techniques, it is particularly advantageous that the 5'endamplification-primer specifically targets the region 30 to 33 in exon 2of the HLA B alleles (according to the numbering given by Zemmour andParham, 1992). According to the sequence data given by these authors,all known alleles which can be determined according to the presentinvention have the following sequence at position 30 to 33 in exon 2:5'-GCCA-3'. Hence, an amplification-primer ending on this sequence willamplify all alleles wanted, and will simultaneously exclude theamplification of exon 2 of many other HLA B-alleles (which arecharacterized by the corresponding sequence 5'-TCCG-3' at position 30 to33 of exon 2) hereby considerably simplifying the DNA typing approach.HLA-B exon 2 alleles of which the DNA-sequence is known (Zemmour andParham, 1992) and which are characterized by the sequence 5'-GCCA-3' atposition 30 to 33 are listed in Table 1.

The invention thus relates to a method for typing or subtyping one ormore HLA-B alleles in a sample characterized by the sequence 5'-GCCA-3'at position 30 to 33 of exon 2 of said HLA-B allele (with said numberingbeing according to Zemmour and Parham, 1992), and more particularly amethod for discriminating HLA-B types which are serologically difficultto discriminate such as for instance B54(22), B52(5), B7801, B62(15),B75(15), B71(70), B72(70), B46, B79, B53, B5102, B5103 and B58(17), withsaid method comprising at least the following steps:

(i) possibly extracting sample nucleic acid,

(ii) amplifying the nucleic acid of HLA-B alleles characterized by thesequence 5'-GCCA-3' at position 30 to 33 of exon 2 of said HLA-B allelewith at least one 5' end amplification primer selected from thefollowing list:

    5'-AGGTATTTCTACACCGCCA-3'                                  (B25P, SEQ ID NO 1)

or sequence variants thereof, such as:

    5'-AGGTATTTCCACACCGCCA-3'                                  (SEQ ID NO 2)

    5'-AGGTATTTCGACACCGCCA-3'                                  (SEQ ID NO 3)

or other sequence variants, with said sequence variants containingdeletions, and/or insertions, and/or substitutions of one or morenucleotides provided that the 3' end GCCA sequence is retained and thatthese sequence variants can be caused to specifically amplify the sameHLA-B alleles as the B25P primer or variants thereof as designatedabove,

in combination with an appropriate 3' end primer being chosen from thesame alleles as the above defined 5' end primers,

with said 5' and 3' end primers being possibly labeled; and,

(iii) hybridizing the amplified product, being possibly labeled duringor after amplification, at appropriate conditions with one or moresuitable probes selected from region 15 to 261 of the HLA-B exon 2region, with said numbering being according to Zemmour and Parham, 1992,

(iv) washing at appropriate washing conditions,

(v) detecting the hybrids formed; and,

(vi) inferring the allele present from the observed hybridizationpattern.

In order to perform the process of the invention as illustrated above,it may be necessary to perform an extraction of sample nucleic acidaccording to any of the techniques known in the art. In case ofextraction of RNA, generation of cDNA is necessary; otherwise cDNA orgenomic DNA is extracted.

The term "primer" refers to a single stranded DNA oligonucleotidesequence or specific primer (SPs) capable of acting as a point ofinitiation for synthesis of a primer extension product which iscomplementary to the nucleic acid strand to be copied. The length andthe sequence of the primer must be such that they allow to prime thesynthesis of extension products. Preferably the primer is about 5-50nucleotides, more preferably from about 10 to 21 nucleotides. Specificlength and sequence of the primer will depend on the complexity of therequired DNA or RNA targets, as well as on the conditions of primer usesuch as temperature and ionic strength.

The fact that amplification-primers do not have to match exactly withthe corresponding template sequence to warrant proper amplification,providing that an exact match at the last three nucleotides at the 3'end of the primer is maintained, is amply documented in the literature(Kwok et al., Nucleic Acids Research 18:999-1005, 1990; Sommer andTautz, Nucleic Acids Research 17, 6749, 1989).

The term "probe" refers to single stranded sequence-specificoligonucleotides. (SSO's) which have a sequence which is exactlycomplementary to the target sequence of the allele to be detected.

Preferably, these probes are about 5 to 50 nucleotides long, morepreferably from about 10 to 18 nucleotides.

The expressions "appropriate" hybridization and washing conditionsrefers to the fact that in most cases said probes are to hybridize onlyto exactly complementary sequences. Such conditions are exampliflied inthe Examples section. For instance for probe 17 preferred hybridizationand wash temperatures are respectively 54° C. and 58° C. if 3M TMAC isused as hybridization and wash solution. In general, the hybridizationconditions are to be stringent as known in the art (f.i. Maniatis etal., Molecular Cloning: A Laboratory Manual, New York, Cold SpringHarbor Laboratory, 1982).

However, according to the hybridization solution (SSC, SSPE, etc.),these probes should be hybridized at their appropriate temperature inorder to attain sufficient specificity (in most cases differences at thelevel of one point mutation should be discriminated). Theabove-mentioned 5' end primers are referred as B25P primers.

The term "sample" refers to any source of biological material, forinstance blood stains, hair, epithelial cells or peripheral blood cells.Typical samples may include peripheral blood mononuclear cells(PBMNC's), lymphoblastoid cell lines (LCL's), hair cells or the like.The preferred isolated nucleic acid will be genomic DNA. However,cytoplasmic, cellular and poly(A)+RNA may also be used.

The expression "inferring the allele present from the observedhybridization pattern" refers to the central feature of the HLA-B typingmethod of the present invention which involves the identification (alsoreferred to as determination or discrimination) of the HLA-B allelespresent in the sample by analyzing the pattern of binding of a panel ofoligonucleotide probes. Although single probes may also provide usefulinformation, the variation of the HLA-B alleles is dispersed in nature,so rarely is any one probe able to identify uniquely a specific variant.Rather, as shown in the Examples, the identity of an allele is inferredfrom the pattern of binding of a panel of oligonucleotide probes, whichare specific for different segments of the different HLA-B alleles.Depending on the choice of these oligonucleotide probes, each knownallele will correspond to a specific hybridization pattern upon use of aspecific combination of probes. Each allele will also be able to bediscriminated from any other allele amplified with the same primersdepending on the choice of the oligonucleotide probes. Comparison of thegenerated pattern of positively hybridizing probes for a samplecontaining one or more unkown HLA-B allele to a scheme of expectedhybridization patterns as for instance shown in Table 1, allows one toclearly infer the HLA-B alleles present in said sample.

Since the targeted alleles differ somewhat in sequence at the 3'-end ofexon 2 (Zemmour and Parham, 1992), different 3' end primers should becombined with a 5' end primer (B25P or variants thereof) to accomplishspecific amplification of all alleles of interest.

The present invention thus relates to a method as defined above,characterized further in that at least one of the following 3' endamplification primers (B23) is used:

    5'-TCTGGTTGTAGTAGCCGCGCA-3'                                (B23P1, SEQ ID NO 4),

or sequence variants thereof, such as:

    5'-TCTGGTTGTAGTAGCGGAGCG-3'                                (B23P2, SEQ ID NO 5),

    5'-TCCGCAGGTTCTCTCGGTA-3'                                  (B23P3, SEQ ID NO 6),

or other sequence variants, with said sequence variants containingdeletions, and/or insertions, and/or substitutions of one or morenucleotides provided that these sequence variants can be caused tospecifically amplify the same HLA-B alleles as the B23P1 primer orvariants B23P2 or B23P3 as designated above, with said primers beingpossibly provided with a detectable label, such as biotin.

As indicated, these primers will be further referred to as B23P1, B23P2,and B23P3, respectively. Primers B23P1 and B23P2 target the region 241to 261 in HLA B exon 2; primer B23P3 targets the region 219 to 237(numbering according to Zemmour and Parham, 1992). The alleles of whichthe DNA-sequence corresponds completely with the 3'end of the primersmentioned above, are identified in Table 1.

From these data, it can be concluded that in order to achieveamplification of all HLA-B alleles of interest, the amplification withprimer set

    B25P/B23P1

should at least be combined with one of the following sets:

    B25P/B23P2

or,

    B25P/B23P3.

From the data given in Table 1, it can also be concluded that somealleles are amplified exclusively with one primer set and not with oneof the other combinations set forward. E.g. among other alleles, B*4601or B*7801 are only amplified with primer set B25P/B23P1. Whereas forinstance alleles B*1503 or B*5801 are amplified with the primersetsB25P/B23P1 and B25P/B23P3, and B25P/B23P2 and B25P/B23P3 respectively.It should also be mentioned that from the available sequence data(Zemmour and Parham, 1992) other 3' end amplification primers may beselected with which amplification of a particular part of HLA-B exon 2alleles may be achieved if combined with 5'-end primer B25P or variantsthereof.

In one embodiment of the invention, amplification with the two primersets of choice is performed in different reaction tubes and theamplified products are hybridized separately as indicated in the typingscheme in FIG. 1.

In another embodiment of the invention, amplification with the twoprimer sets of choice is performed in different reaction tubes and theamplified products are mixed after amplification and hybridizedtogether.

In a preferred embodiment of the invention, the different primersinvolved (either B25P, B23P1 and B23P2 or B25P, B23P1 and B23P3) aremixed and amplification is performed in a single reaction tube afterwhich the amplified products are hybridized together.

The amplification method used can be either PCR (Saiki et al., Science239:487-491, 1988), nucleic acid sequence-based amplification (NASBA,Guateli et al., PNAS (USA);87:1874-1878, 1990; Compton,Nature;350:91-92, 1991), transcription-based amplification system TAS,Kwoh et al., Proc. Natl. Acad. Sci. (USA) 86:1173-1177, 1989), stranddisplacement amplification (SDA, Duck et al., Biotechniques 9:142-147,1990; Walker et al. Proc. Natl. Acad. Sci. (USA) 89:392-396, 1992),amplification by means of Qβ replicase (Lizardi et al., Bio/Technology6:1197-1202, 1988; Lomeli et al., Clin. Chem. 35:1826-1831, 1989) or anyother suitable method liable to amplify nucleic acid molecules usingprimer extension. During amplification, the amplified products can beconveniently labeled either using labeled primers or by incorporatinglabeled nucleotides. Labels may be isotopic (³² P, ³⁵ S, etc.) ornon-isotopic (biotin, digoxigenin, etc.).

In order to distinguish the amplified alleles from each other, theamplification products are hybridized to a set of sequence-specific DNAprobes (also referred to as SSOs) targetting HLA-B exon 2 regionslocated in between the amplification primer regions chosen. Differenthybridization formats can be used such as the conventional dot-blotformat, sandwich hybridization or reverse hybridization (such as thereversed dot blot format). A particular set of DNA probes was selectedwhich enables to distinguish the alleles of interest from each other andfrom other alleles described, whether or not these alleles are presentin the homozygous or heterozygous state.

For this purpose, 20 basic DNA probe sequences were identified which aredesigned to be functional in TMAC (tetramethylamonium chloride)hybridization and wash conditions as illustrated in the Examplessection. Most of these probes target the most variable regions of HLA-Bexon 2 and can be caused to hybridize to more than one HLA-B allele.Some probes were selected because they are allele-specific; these probesare probe 8 (SEQ ID NO 14), 12 (SEQ ID NO 18), and 16 (SEQ ID NO 22),which exclusively hybridize to B*5401, B*4601, and B*7901 respectively.The regions targeted by the different probes are schematicallyrepresented in FIG. 2. In Table 2, the sequences of the probes aregiven. An interpretation is given in Table 1. Further on, the same 20basic probes or variants thereof were tested in hybridization and washconditions specific for SSPE as reviewed in Example 5 and Table 2 bis.As can be seen in Table 2 bis, 9 of the originally TMAC-tested probesand some of the new variant probes are particularly preferred aboveothers under the specific SSPE buffer conditions used.

The present invention thus relates to a method as defined above, whereinone or more hybridization probes are selected from Table 2 (SEQ ID NO 7to 26), or sequence variants thereof, with said sequence variantscontaining deletions and/or insertions of one or more nucleotides,mainly at their extremities (either 3' or 5'), or substitutions of somenon-essential nucleotides (i.e. nucleotides not essential todiscriminate between alleles) by others (including modified nucleotidessuch as inosine), or with said variants consisting of the complement ofany of the above-mentioned oligonucleotide probes, or with said variantsconsisting of ribonucleotides instead of deoxyribonucleotides, allprovided that said variant probes can be caused to hybridize with thesame specificity as the oligonucleotide probes from which they arederived.

The latter implies that variants contemplated within this aspect of thepresent invention can be defined as probes hybridizing with the samespecificity as the probe they are derived from under different, butstringent, hybridization and wash conditions (different solutions,different concentrations of buffer, different concentrations of probe,different temperatures). Such variants are f.i. contained within thesequences given in SEQ ID NO 27 to 52 (Table 2 bis).

According to the hybridization solution (SSC, SSPE, TMAC, etc.), theseprobes should be stringently hybridized at their appropriate temperaturein order to attain sufficient specificity (in most cases differences atthe level of one point mutation should be discriminated). However, byslightly modifying the DNA probes listed in Table 2, either by adding ordeleting one or a few nucleotides at their extremities (either 3' or5'), or substituting some non-essential nucleotides (i.e. nucleotidesnot essential to discriminate between alleles) by others (includingmodified nucleotides such as inosine) these probes or variants thereofcan be caused to hybridize specifically at the same hybridizationconditions (i.e. the same temperature and the same hybridizationsolution). Also changing the amount (concentration) of probe usedrelevant to each other may be beneficial to obtain more specifichybridization results. It should be noted in this context, that incontradiction to SSPE based buffer solutions, probes of the same length,regardless of their GC content, will hybridize specifically atapproximately the same temperature in TMAC solutions (Jacobs et al.,Nucleic Acids Research 16:4637-4650, 1988).

Preferred basic and variant probes using different hybridization andwash buffer conditions are illustrated in the Examples section of thepresent invention. Some of these preferred probes are included in SEQ IDNO 7 to 52.

Among the alleles which are the subject of this invention, B*1501 andB*1504 cannot be discriminated from each other since these sequences areexactly the same in the second exon. Differences between both allelesare found in the 5' end of exon 3 (Zemmour and Parham, 1992). For thesame reason B*5101 to B*5104 cannot be distinguished. Also for thesealleles discrimination can be achieved in exon 3 (Zemmour and Parham,1992). Thus a more complete typing system will include a primer andprobe combination to discriminate between types in exon 3.

The above-mentioned DNA typing methods in which primer pair B25P/B23P2is used and hybridization is observed with SSO's 7 (SEQ ID NO 13) and 9(SEQ ID NO 15) are preferably carried out additionally and separatelywith primer pair B25P and the B23P1 and/or B23P3 primers. For the latterapplication, a primer with the following sequence:

    5'-GACGACACG/CCT/AGTTCGTGA-3'                              B25PX1 (SEQ ID NO 53)

can be used as alternative for the B25P primer.

As detailed in the Examples section, this additional amplification steprules out the possibility that co-amplification of the HLA-AR pseudogene(when primer B23P2 is used) occurs, and thus allows for a more accurateHLA-B typing procedure.

Suitable assay methods for purposes of the present invention to detecthybrids formed between the oligonucleotide probes and the nucleic acidsequences in a sample may comprise any of the assay formats kown in theart. For example, the detection can be accomplished using a dot blotformat, the unlabelled amplified sample being bound to a membrane, themembrane being incorporated with at least one labelled probe undersuitable hybridization and wash conditions, and the presence of boundprobe being monitored. Probes can be labelled with radioisotopes or withlabels allowing chromogenic or chemilumeniscent detection such ashorse-radish peroxidase coupled probes.

An alternative is a "reverse" dot-blot format, in which the amplifiedsequence contains a label. In this format, the unlabelledoligonucleotide probes are bound to a solid support and exposed to thelabelled sample under appropriate stringent hybridization and subsequentwashing conditions. It is to be understood that also any other assaymethod which relies on the formation of a hybrid between the nucleicacids of the sample and the oligonucleotide probes according to thepresent invention may be used.

According to an advantageous embodiment, the process of typing of HLA-Balleles contained in a biological sample comprises the steps ofcontacting amplified copies derived from the genetic material, with asolid support on which probes as defined above, have been previouslyimmobilized.

The term "solid support" can refer to any substrate to which anoligonucleotide probe can be coupled, provided that it retains itshybridization characteristics and provided that the background level ofhybridization remains low. Usually the solid substrate will be amicrotiter plate, a membrane (e.g. nylon or nitrocellulose) or amicrosphere (bead). Prior to application to the membrane or fixation itmay be convenient to modify the nucleic acid probe in order tofacilitate fixation or improve the hybridization efficiency. Suchmodifications may encompass homopolymer tailing, coupling with differentreactive groups such as aliphatic groups, NH₂ groups, SH groups,carboxylic groups, or coupling with biotin or haptens.

According to another advantageous embodiment, the process of typing ofHLA-B alleles contained in a biological sample comprises the steps ofcontacting amplified copies derived from the genetic material, witholigonucleotide probes which have been immobilized as parallel lines ona solid support.

According to this preferred embodiment of the invention, one or more ofthe above defined probes are used for immobilization and incorporationinto a reverse phase hybridization assay, preferably for immobilizationas parallel lines onto a solid support such as a membrane strip, fortyping of HLA-B alleles according to a method as defined above.According to this advantageous method, the probes are immobilized in aLine Probe Assay (LiPA) format. This is a reverse hybridization format(Saiki et al., PNAS (USA); 86:6230-6234, 1989) using membrane strips onwhich 20 or more oligonucleotide-probes (including negative or positivecontrol oligonucleotides) are conveniently applied as parallel lines.The LiPA strips are prepared as described by Stuyver et al. (J. Gen.Virol. 74: 1093-1102, 1993).

The invention thus also relates to a solid support, preferably amembrane strip, carrying on its surface, one or more probes as definedabove, coupled to the support in the form of parallel lines.

The LiPA is a very rapid and user-friendly hybridization test. Resultscan be read 4 h. after the start of the amplification. Afteramplifiaction during which usually a non-isotopic label is incorporatedin the amplified product, and alkaline denaturation, the amplifiedproduct is contacted with the probes on the membrane and thehybridization is carried out for about 1 to 1.5 h. After a brief wash(10 to 30 min.) the detection procedure is started. All these steps arecarried out in the same hybridization recipients, hereby minimalizinghands-on time. From the hybridization pattern generated, the HLA-Ballele(s) present can be deduced either visually but preferably usingdedicated software. The LiPA format is completely compatible withcommercially available scanning devices, thus rendering automaticinterpretation of the results very reliable. All those advantageous makethe LiPA format liable for the use of HLA typing in a routine setting.The LiPA format should be particularly advantageous for typing thosealleles which are difficult to type by routine serological means.

The present invention also relates to a method for detecting andidentifying novel HLA-B alleles, different from the known HLA-B alleles,comprising the steps of:

determining which HLA-B allele(s) the is(are) present in a biologicalsample, according to the process as defined above,

in the case of observing a sample which does not generate ahybridization pattern compatible with those defined in Table 2,sequencing the portion of the HLA-B exon 2 sequence corresponding to theaberrantly hybridizing probe of the new HLA-B allele to be determined.

The probe set of the present invention does not only allow one todiscriminate the alleles of which the sequences are known but alsoalleles with as yet unknown sequences can be detected as is exemplifiedin Example 3. In this example it is shown that B71 can be distinguishedfrom B72 (B*1503) using probe 13. This is particularly advantageoussince discrimination of B71 and B72 on serological basis is problematic.

The invention thus relates to a method for discriminating between B72(B*1503) and non-B72 HLA-B alleles of the B70 group (B70, B71, i.e.alleles different from B*1503), using the method as defined above, withsaid non-B72 HLA alleles being characterized by the fact that they donot form a hybrid with at least one of the following probes whichhybridize to the B*1503 allele (f.i. probe 13 (SEQ ID NO 19), probe 7(SEQ ID NO 13), probe 10 (SEQ ID NO 16), probe 18 (SEQ ID NO 24), andprobe 19 (SEQ ID NO 25)).

Once the sequence of these new alleles become available, new probes canbe deduced which allow for a specific detection of these new alleles.The addition of these probes to the set of 20 basic probes listed inTable 1 will thus improve the level of discrimination and the relevanceof this typing procedure.

The present invention thus also relates to a novel HLA-B alleleccrresponding to an as yet, at the nucleic acid sequence levelunidetermined B70 HLA-B type, determined according to the above definedmethod, with said allele being characterized by the fact that it forms ahybrid with probe 2 (SEQ ID NO 8, see Table 2), whilst not forming ahybrid with probe 13 (SEQ ID NO 19, see Table 2), and with said allelebeing different from the HLA-B B70 allele B*1503 in at least onenucleotide position in the region spanning nucleotides 192 to 209, withsaid numbering being according to Zemmour and Parham, 1992.

The present invention also relates to a composition comprising at leastone of the oligonucleotide amplification primers selected from thefollowing list:

    5'-AGGTATTTCTACACCGCCA-3'                                  (B25P, SEQ ID NO 1)

or sequence variants thereof, such as:

    5'-AGGTATTTCCACACCGCCA-3'                                  (SEQ ID NO 2)

    5'-AGGTATTTCGACACCGCCA-3'                                  (SEQ ID NO 3)

or other sequence variants thereof, with said sequence variantscontaining deletions and/or insertions and/or substitutions of one ormore nucleotides provided that the 3' end GCCA sequence is retained andthat these sequence variants can be caused to specifically amplif thesame HLA-B alleles as the B25P primer or variants thereof as designatedabove,

    5'-TCTGGTTGTAGTAGCCGCGCA-3'                                (B23P1, SEQ ID NO 4),

or other sequence variants thereof, such as:

    5'-TCTGGTTGTAGTAGCGGAGCG-3'                                (B23P2, SEQ ID NO 5),

    5'-TCCGCAGGTTCTCTCGGTA-3'                                  (B23P3, SEQ ID NO 6),

or sequence variants thereof, with said sequence variants containingdeletions and/or insertions and/or substitutions of one or morenucleotides provided that sequence variants can be caused tospecifically amplify the same HLA-B alleles as the B23P1 primer orvariants B23P2 or B23P3 thereof as designated above, with said primersbeing possibly provided with a detectable label, such as biotin, andwith set primers being possibly immobilized onto a solid support.

Preferably such compositions contain at least two or more amplificationprimers selected from said list. More preferably the amplificationprimer set B25P/B23P1 is combined with at least one of the followingsets:

    B25P/B23P2, or B25P/B23P3.

In addition to the above-mentioned components, said composition may alsocomprise at least one of the following primers:

    5'-GACGACACG/CCT/AGTTCGTGA-3' B25PX1                       (SEQ ID NO 53)

The present invention also relates to a composition comprising at leastone oligonucleotide probe selected from the following list of probes:

SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ IDNO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, or SEQ ID NO 26, (as givenin Tables 2),

or sequence variants thereof, with said sequence variants containingdeletions and/or insertions of one or more nucleotides, mainly at theirextremities (either 3' or 5'), or substitutions of some non-essentialnucleotides (i.e. nucleotides not essential to discriminate betweenalleles) by others (including modified nucleotides such as inosine), orwith said sequence variants consisting of the complement of any of theabove-mentioned oligonucleotide probes, or with said sequence variantsconsisting of ribonucleotides instead of deoxyribonucleotides, allprovided that said variant probes can be caused to hybridize with thesame specificity as the oligonucleotide probes from which they arederived. Such variants may f.i. be chosen from the following list ofprobes:

SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31,SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36,SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41,SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46,SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, orSEQ ID NO 52 (as given in Table 2 bis).

Preferably such compositions contain at least two, three or more ofthese probes.

The present invention also relates to a kit for typing at least oneHLA-B allele from a biological sample liable to contain it, comprisingthe following components:

when appropriate at least one amplification primer chosen among any ofthose defined above,

at least one probe, with said probe(s) being preferentially immobilizedon a solid substrate, and more preferentially on one and the samemembrane strip, and with said probe(s) being selected among any of thosedefined above,

a buffer or components necessary for producing the buffer enablinghybridization reaction between these probes and the amplified product tobe carried out;

when appropriate, a means for detecting the hybrids resulting from thepreceding hybridization.

The present invention relates also to a kit for typing of at least oneHLA-B allele from a biological sample liable to contain it, moreparticularly of a sample liable to contain serologically difficult todiscriminate HLA-B types, subsequent to amplification of the nucleotidesencoding the HLA-B alleles present in said sample, using one or moreprimer set combination according to a method as defined above,comprising:

at least one probe, with said probe(s) being preferentially immobilizedon a solid substrate, and more preferentially on one and the samemembrane strip, with said probe(s) being selected among any of thosedefined above,

a buffer or components necessary for producing the buffer enablinghybridization reaction between these probes and the amplified product tobe carried out;

means for detecting the hybrids resulting from the precedinghybridization,

possibly also including an automated scanning and interpretation devicefor interpretating the results and inferring the allel present from theobserved hybridization pattern.

FIGURE AND TABLE LEGENDS

FIG. 1 and FIG. 1A: Schematic representation of the typing approachaccording to the present invention.

FIG. 2: Localization of the primers and probes of the present inventionon an axis representing exon 2 of the HLA-B gene. The numbering isaccording to Zemmour and Parham, 1992.

FIG. 3: Amplification (top panel) and dot-blot hybridization (bottompanel) results obtained with 13 patient-samples and 7 controls. Thematerial was amplified using primer set B25P/B23P1, dot-blotted, andhybridized with probes 6 (SEQ ID NO 12) and 17 (SEQ ID NO 23) asdescribed in examples 1 and 2.

FIG. 4: Dot-blot hybridization results obtained for 15 samples (n* 1 to15), among which samples harbouring B70 variant alleles and B46 alleles.The amplified material was applied to nylon membranes. Subsequentlythese membranes were hybridized with SSO-probes 13 (SEQ ID NO 19), 12(SEQ ID NO 18), 15 (SEQ ID NO 21), 6 (SEQ ID NO 12), and 2 (SEQ ID NO 8)as indicated. The results are discussed in examples SSO 3 and 4 andsummarized in Table 3.

Table 1: Amplified HLA-B alleles (exon 2) with the primer sets of theinvention. Also the presence of the 3' end primer sequence in thealleles and the hybridization pattern with the set of the 19 SSO-probesis indicated.

Table 2: Panel of typing SSO-probes for HLA-B alleles.

Table 2 bis: Panel of typing SSO-probes for HLA-B alleles tested to beused in a reverse hybridization (Line Probe Assay, LIPA) format. All ofthese probes are derived from the basic set of 20 SSOs given in Table 2.The preferred probes are indicated as "+".

Table 3: Summary of the hybridization results obtained with 15 amplifiedsamples hybridized with 5 SSO-probes as described in examples 3 and 4.See also FIG. 4.

Table 4: Hybridization results obtained with a LiPA strip onto which 20oligonucleotide probes were immobilized. Positive hybridization signalsafter hybridization with material obtained respectively afteramplification 1 and 2 (see Example 6) are indicated as "+".

ABBREVIATIONS

TMAC: Tetramethylamoniumchloride

SSO: Sequence-specific oligonucleotide

DIG 11-ddUTP: Digoxigenin-11-2',3'-dideoxy-uridine-5'-triphosphate

DIG: Digoxigenin

AMPPD: 3-(2'-spiroadamantane)4methoxy-4-(3"-phosphoryloxy)-phenyl-1'2-dioxetane

AP: alkaline phosphatase

EXAMPLES Example 1 Specific Amplification of Certain HLA-B Alleles withPrimer Set B25P/B23P1

By way of example the amplification--specificity with one of the primersets is illustrated. As indicated in FIG. 3, 12 samples and 7 controlswere amplified with primer-set B25P/B23P1 (SEQ ID NO 1 and 4). Startingfrom cell-material, genomic DNA was prepared by standard protocols.Approximately 0.5 μg of genomic DNA was mixed with PCR buffer containing12.5 pmoles of each primer; 200 mM of each dNTP (Pharmacia LKBBiotechnology, Uppsala, Sweden); 10 mM Tris HCl (pH 8.5); 50 mM KCl; 1mM MgCl₂ ; 0.01% gelatine; 0.025% NP-40 and 1 Unit of Taq (Thermusaquaticus) DNA polymerase (Boerhinger, Mannheim GmbH, FRG) adjusted to afinal volume of 50 μl with double distilled water. Samples were heatedat 95° C. for 10 min and subjected to 35 cycles of PCR, each consistingof 94° C. for 1 min, 55° C. for 30 sec, 72° C. for 1 min with a 10 min72° C. final extension in a DNA Thermal Cycler (Techne and Perkin-ElmerCetus Corp., Norwalk, Conn.). The amplification products werecharacterized by a 1.5% agarose gel electrophoresis.

The results are shown in FIG. 3. In all samples in which amplifiablealleles were present a distinct band of about 246 basepairs, aspredicted by the available sequence data, was observed afterethidiumbromide staining.

Samples 1, 4, 5, 6, 8, 9, 10, 11, and 12 harbour the B*7801 allele(indicated in FIG. 1 as BSNA, BX1 or BTe76). In sample 2 and 3 andcontrol 15 a B8 allele (B*0801) is found. Samples 16 to 19 arehomozygous controls for B35, B55, B56 and B54 respectively. In thenegative controls (13 and 14 harbouring respectively the B*3701 andB*1801 alleles) no band could be observed. These alleles have the5'-TCCG-3' sequence at position 30 to 33 of exon 2 instead of 5'-GCCA-3'(Zemmour and Parham, 1992).

Example 2 Dot Blot Typing Assay for Detection of the B78 Allele(s)

In this example the specific typing of the B*7801 allele is describedmaking use of probes 6 and 17 (SEQ ID NO 12 and 23). Since B*7801 is theonly allele hybridizing with both probes (6 and 17), B*7801 can bediscriminated from the other alleles amplified with primer setB25P/B23P1.

The sequence-specific oligonucleotide-probes (SSOs) were chemicallysynthesized and labelled at their 3' end withdigoxigenin-11-2'3'-dideoxy-uridine-5'-triphosphate (DIG-11-ddUTP) andDNA deoxynucleotidylexotransferase. Thirteen samples and 7 controls wereamplified with primerset B25P/B23P1 as described in example 1.

Consequently 2 μl of PCR products were dot blotted onto nylon membranes(Hybond N Plus, Amersham, Buckinghamshire, UK) denatured by soaking thefilters in 0.4N NaOH for 5 min and neutralized in 10 ml 10× SSPE (salinesodium phosphate EDTA) for 15 min. After blotting, membranes wereprehybridized at 54° C. for 30 min in 10 ml of hybridization solutioncontaining 50 mM Tris-HCl, (pH 8.0), 0.1% SDS, 2 mM EDTA, 3M TMAC(tetramethylammonium chloride, Janssen Chimica, Geel, Belgium). Forhybridization, 3 pmol/ml DIG-labelled SSO were added to theprehybridization solution for 1 hour at 54° C., except for SSO-6 (SEQ IDNO 12) (52° C.). To remove probe excess, the filters were washed twicein (2× SSPE, 0.1% SDS) at room temperature for 10 min and then inhybridization solution for 15 min at the stringent temperature 58° C.,except for the SSO-6 which was washed at 54° C.

The non-isotopic detection was performed using anti-DIG alkalinephosphatase, Fab fragments (anti-DIG-AP) and vizualization was obtainedwith the chemiluminescent substate AMPPD (3-(2'-spiroadamantane)-4methoxy-4-(3"-phosphoryloxy)-phenyl-1,2-dioxetane). The drainedmembranes were exposed to an X-ray film (X-omat AR5, Kodak) in acassette for 15-30 min. All reagents used in this oligotyping procedurewere purchased form Boerhinger, Mannheim GmH, FRG. The results areillustrated in FIG. 3. All samples in which an allele of the B78 type ispresent (BSNA, BX1 or BTe76) clearly hybridize with probes 6 and 17 (SEQID NO 12 and 23). Other samples (n* 2, 3, 7, 15, 16, 17, 18, and 19)either hybridize with probe 17 (SEQ ID NO 23) or probe 6 (SEQ ID NO 12)but not with both. Since samples 13 and 14 are not amplified with theprimerset used, hybridization signals are observed with neither probe 6(SEQ ID NO 12) nor probe 17 (SEQ ID NO 23).

Example 3 Typing and Subtyping of B70 Variants

In this example the typing and subtyping of B70 variants is illustratedusing 7 samples harbouring B70 variants. Also a blank and 7 non-B70samples were included as controls (see table 3). B71 and B72 are allelessubtypic to the broad antigen HLA-B70. The sequence of the B72 allele(B*1503) is published (Zemmour and Parham, 1992); up to recently the B71sequence was not. No monospecific B71 and B72 reagents are described andthe presence of other antigens, most prominently B35 and B62, obscureexact serological assignment of the B70 variants. Distinction betweenB70 variants can be made by isoelectric focusing, but this is not afavourable technique in a routine setting.

By using the primer set B25P/B23P1 (SEQ ID NO 1 and 4) and a combinationof some probes of the invention the B72 variants could be distinguishedfrom other B70 variants. Here it should be emphasized that othervariants than B71 and B72, as yet unidentified, might exist.

From a set of samples, known to harbour B70 variant alleles (samples 1to 6 and 8, in table 3) DNA was extracted and amplified as described inexample 1. The amplified products were applied to 5 nylon membraneswhich were further processed as described in example 2. These membraneswere hybridizied respectively with the following sets of probes: 2, 6,12, 13 and 15 (SEQ ID NO 8, 12, 18, 19 and 21). The results obtainedwith these probes are shown in FIG. 4. These results are summarized intable 3. In all cases the B72 variant (B*1503), which was present in 4of the 7 B70 samples, could be distinguished from B71 or other possiblevariants. B*1503 (B72) is characterized by hybriding with probes 2 and13 (SEQ ID NO 8 and 19) while other B70 variants were found to benon-reactive with probe 13 (SEQ ID NO 19). This example, thus, clearlyillustrates the possibility to discriminate between alleles even whenthe sequence-information is not available.

Example 4 Typing of B46 Variants

Two B46 containing samples were typed using the same methods, primerset,and probe combination as discribed in example 3. In FIG. 4 and table 3the typing results are given. By virtue of the presence of probe 12 (SEQID NO 18) in the probe panel, B*4601 can be easily traced andunequivocally distinguished from other alleles. From all allelesamplified by the primer set B25P/B23P1 (SEQ ID NO 1/4), B*4601 is theonly allele hybridizing with probe 12 (SEQ ID NO 18).

Example 5 Line Probe Assay (LiPA) and SSO's for Typing of HLA-B Alleles

The prefered hybridization and wash- media for the Line Probe Assay(LiPA) are SSPE-based buffer solutions. LiPA strips were preparedessentially as described by Stuyver et al. (J. Gen. Virol. 74:1093-1102, 1993). Since in a LiPA format all probes should reactspecifically under the same hybridization- and wash conditions (the samesalt concentration and temperature) and the thermal melting point ofDNA: DNA-hybrid in SSPE depends on the GC-content and lenght of theprobe, modification of the probes listed in Table 2 is required for someprobes to shift from a TMAC based buffer system to a SSPE based buffersystem.

In order to select the most fitting probes to be used in a LiPA format amultitude of probes (listed in Table 2 bis) were synthesized, tailed attheir 3' extremities using TTP and terminal transferase and immobilisedon a solid support (nitrocellulose membrane). These probes werehybridised with target material using the following hybridization andwash conditions:

hybridization: -5×SSPE/0.5% SDS -55° C.

wash: -2×SSPE/0.1% SDS -55° C.

(1×SSPE is 0.18M NaCl, 0.01M NaH₂ PO₄, 1 mM EDTA (pH 7.2))

The probes exhibiting the best test results with respect to specificityand sensitivity under the above mentioned conditions were selected forfurther use on the LiPA-strips. These probes were scored as positive (+)in Table 2 bis. Only 9 of the 20 probes used in the TMAC buffer system(SEQ ID NO 7b, 11, 12, 13, 18, 19, 21, 23 and 24) could be used withoutmodification in the SSPE-based system.

These results clearly demonstrate that slight modifications of thesequence of the probes used might be of relevance and that meticulousprobe design is essential for the development of a reliable LiPA-test.

Example 6 Typing of More Representative Homozygous Cell-lines Using theLiPA Strips

Due to co-amplification of the HLA-AR pseudogene (when primer B23P2 isused) hybridization results with probes 7 (SEQ ID NO 13) and 9 (SEQ IDNO. 15) are equivocal since sequences corresponding to those of probe 7(SEQ ID NO 13) and 9 (SEQ ID NO 15) are present in the pseudogene.

In order to probe the origin of an eventual positive hybridizationsignal an additional amplification was performed using at least one ofthe B23 primers and a primer with the following sequence:

    5'-GACGACACG/CCT/AGTTCGTGA-3' B25PX1                       (SEQ ID NO 53)

The amplification product obtained was consequently hybridized with astrip onto which at least probe 7 and 9 were immobilized.

A positive hybridization signal for probes 7 and 9 in this assayindicates that probe 7 and/or 9 sequences are present in the HLA-Balleles of the sample analysed and hence probes 7 and/or 9 should bescored positive. A negative result means that these probes should beneglected during the interpretation of the results after the firstamplification since their positive hybridization signal originated fromthe pseudogene and not from the HLA-B gene itself.

Hybridization results obtained with some selected samples (homozygonscell-lines A to G) after separate amplification with primers B25P andB23PI/B23P2 (amplification 1) and with primers B25PX1 and B23P1/B23P2(amplification 2) are summarized in Table 4. These results show that forsample A the positive hybridization signal for probes 7 and 9 originatefrom the HLA-B allele. The same is true for the positive signal obtainedwith probe 9 in samples E and F.

If the results after amplification steps 1 and 2 are combined, thefollowing HLA-B alleles can be deduced with the aid of Table

    ______________________________________           sample                 allele    ______________________________________           A     B*0801           B     B*1501           C     B*4001           D     B*4601           E     B*5101           F     B*5301           G     B*5701    ______________________________________

Essentially the same results are obtained when, for amplification 2, thefollowing primer set is used:

    B25P and B23P1/B23P3

                  TABLE 1    ______________________________________    3'-PRIMER          PROBE    ALLELES           B23P1   B23P2   B23P3 1   2   3   4   5   6   7    ______________________________________    4001/  +               +     +    4501/5001    4901           +       +     +    4101   +               +     +    4701                   +     +    1301/1302      +       +     +               +    4401 to        +       +     +    4403    0801   +                         +                   +    5401   +                                 +    5501/  +                                 +           +    5502/    5601/5602    3501 to           +                                 +       +    3506    5301           +       +                 +       +    5101 to        +       +             +           +    5104    5201           +       +                 +       +    52012          +       +             +           +    7801   +                             +           +    1501/1504           +               +                 +   +    1502   +               +                 +   +    1503   +               +         +                   +    4601   +                                 +   +    7901   +               +         +                   +    5701/5702      +       +                 +   +    5801           +       +                 +       +    ______________________________________    PROBE           8     9     10  11  12  13  14  15  16  17  18                               19  7b    ______________________________________                               4001/ +   +     + + +                               4501/5001                               4901  +   +     +  +                               4101  +   +     + + +                               4701  +   +     +  +                               1301/1302                                   +   +     +                               4401 to                                   +   +     +  +                               4403                               0801 +     +   +  +                               5401                                   + +      +  +  +                               5501/                                   +      +  +  +                               5502/                               5601/5602                               3501 to                                   +     +    + +                               3506                               5301 +     +    +                               5101 to                                   +     +    +                               5104                               5201  +   +     +                               52012 +   +     +                               7801 +     +   +  +                               1501/1504                                   +   +     + +                               1502 +    +     + +                               1503  +   +     + +                               4601  +  +                               7901 +       +  + +                               5701/5702                                   +                               5801   +    ______________________________________

                  TABLE 2    ______________________________________    Oligonucleotide probe DNA sequences    ______________________________________     1      5'-GCTTCATCACCGTGGGCT-3'                                (SEQ ID NO 7)     2      5'-CGCTTCATCTCAGTGGGC-3'                                (SEQ ID NO 8)     3      5'-CGCTTCATTGCAGTGGGC-3'                                (SEQ ID NO 9)     4      5'-CGCTTCATCGCAGTGGGC-3'                                (SEQ ID NO 10)     5      5'-CCGAGGATGGCGCCCCGG-3'                                (SEQ ID NO 11)     6      5'-TCCGAGGACGGAGCCCCG-3'                                (SEQ ID NO 12)     7      5'-AGTCCGAGAGAGGAGCCG-3'                                (SEQ ID NO 13)     7b     5'-GTCCGAGGAAGGAGCCGC-3'                                (SEQ ID NO 26)     8      5'-GCGCCGTGGGTGGAGCAG-3'                                (SEQ ID NO 14)     9      5'-GGGACCGGAACACACAGA-3'                                (SEQ ID NO 15)    10      5'-GGACCGGGAGACACAGAT-3'                                (SEQ ID NO 16)    11      5'-GGACGGGGAGACACGGAA-3'                                (SEQ ID NO 17)    12      5'-ACACAGAAGTACAAGCGC-3'                                (SEQ ID NO 18)    13      5'-CAGATCTCCAAGACCAAC-3'                                (SEQ ID NO 19)    14      5'-ACAGATCTTCAAGACCAA-3'                                (SEQ ID NO 20)    15      5'-CAGATCTACAAGGCCCAG-3'                                (SEQ ID NO 21)    16      5'-ACAGATCTGCAAGACCAA-3'                                (SEQ ID NO 22)    17      5'-ACAGACTGACCGAGAGAG-3'                                (SEQ ID NO 23)    18      5'-CACAGACTTACCGAGAGA-3'                                (SEQ ID NO 24)    19      5'-ACCGAGAGAGCCTGCGGA-3'                                (SEQ ID NO 25)    ______________________________________

                  TABLE 2b    ______________________________________    S01   +     5'-CTTCATCACCGTGGGCT-3'                                     (SEQ ID NO 27)    S02   +     5'-GCTTCATCTCAGTGGGC-3'                                     (SEQ ID NO 28)    S03   +     5'-GCTTCATTGCAGTGGGC-3'                                     (SEQ ID NO 29)    S04   +     5'-CTTCATCGCAGTGGGC-3'                                     (SEQ ID NO 30)    S05         5'-GATGGCGCCCCGG-3'  (SEQ ID NO 31)    S05(2)      5'-GAGGATGGCGCCCCGG-3'                                     (SEQ ID NO 32)    S05(3)          +     5'-CCGAGGATGGCGCCCCGG-3'                                     (SEQ ID NO 11)    S06         5'-GGACGGAGCCCCG-3'  (SEQ ID NO 33)    S06(2)      5'-CGAGGACGGAGCCCCG-3'                                     (SEQ ID NO 34)    S06(3)      5'-CCGAGGACGGAGCCCCG-3'                                     (SEQ ID NO 35)    S06(4)      5'-TCCGAGGACGGAGCCCCG-3'                                     (SEQ ID NO 12)    S06(5)          +     5'-TCCGAGGACGGAGCCCCGG-3'                                     (SEQ ID NO 36)    S07         5'-TCCGAGAGAGGAGCC-3'                                     (SEQ ID NO 37)    S07(2)      5'-AGTCCGAGAGAGGAGCC-3'                                     (SEQ ID NO 38)    S07(3)          +     5'-AGTCCGAGAGAGGAGCCG-3'                                     (SEQ ID NO 13)    S07b  +     5'-GTCCGAGGAAGGAGCCGC-3'                                     (SEQ ID NO 26)    S08   +     5'-CCGTGGGTGGAGCAG-3'                                     (SEQ ID NO 39)    S08(2)      5'-CGCCGTGGGTGGAGCAG-3'                                     (SEQ ID NO 40)    S09         5'-GGACCGGAACACACAGA-3'                                     (SEQ ID NO 41)    S09(2)      5'-GACCGGAACACACAGA-3'                                     (SEQ ID NO 42)    S09(3)      5'-GACCGGAACACACAG-3'                                     (SEQ ID NO 43)    S09(4)          +     5'-GGACCGGAACACACAG-3'                                     (SEQ ID NO 44)    S10   +     5'-GACCGGGAGACACAGAT-3'                                     (SEQ ID NO 45)    S11   +     5'-ACGGGGAGACACGGAA-3'                                     (SEQ ID NO 46)    S12   +     5'-ACACAGAAGTACAAGCGC-3'                                     (SEQ ID NO 18)    S13   +     5'-CAGATCTCCAAGACCAAC-3'                                     (SEQ ID NO 19)    S14         5'-CACAGATCTTCAAGACCAAC-3'                                     (SEQ ID NO 47)    S14(2)      5'-ACAGATCTTCAAGACCAAC-3'                                     (SEQ ID NO 48)    S14(3)          +     5'-CAGATCTTCAAGACCAA-3'                                     (SEQ ID NO 49)    S15   +     5'-CAGATCTACAAGGCCCAG-3'                                     (SEQ ID NO 21)    S16   +     5'-CACAGATCTGCAAGACCAA-3'                                     (SEQ ID NO 50)    S17   +     5'-ACAGACTGACCGAGAGAG-3'                                     (SEQ ID NO 23)    S18   +     5'-CACAGACTTACCGAGAGA-3'                                     (SEQ ID NO 24)    S19         5'-CGAGAGAGCCTGCGG-3'                                     (SEQ ID NO 51)    S19(2)          +     5'-CGAGAGAGCCTGCGGA-3'                                     (SEQ ID NO 52)    ______________________________________

                  TABLE 3    ______________________________________            PROBE    SAMPLE    2     12    15  6    13  B70 or B46 allele identified    ______________________________________     1  B70/B78   +             +    +   B*1503     2  B51/B70   +                      B70 (B71?)     3  B49/B71   +                      B70 (B71?)     4  B07/B72   +                  +   B*1503     5  B49/B72   +                  +   B*1503     6  B07/B72   +                  +   B*1503     7  B07/B49                          --     8  ?/B70     +                      B70 (B71?)     9  blank                            --    10  B75/B46         +            +   B*4601    11  B46/B46         +                B*4601    12  B51/B35                 +        --    13  B08/B35   +                      --    14  B08/B44                 +        --    15  B44/B44                          --    ______________________________________

                                      TABLE 4    __________________________________________________________________________                                                Amplifi-    Amplification 1                             cation 2    Samples        1 2 3 4 5 6 7 7b                        8 9 10                              11                                12                                  13                                    14                                      15                                        16                                          17                                            18                                              19                                                7 9    __________________________________________________________________________    A     +         +     +         +     +   + + +    B         + +   +     + +     +               +                                                  +    C   +             +   + +     +               +                                                  +    D         + +         + +   +    E       +     + +     +         +             +                                                  +    F         +   + +     +         +             +                                                  +    G         + +         + +   +    __________________________________________________________________________

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 53    (2) INFORMATION FOR SEQ ID NO: 1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Primer B25P    (B) LOCATION: anneals to nucleotides 15-33 of exon 2 of e.g.    HLA-B*3501    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:    AGGTATTTCTACACCGCCA19    (2) INFORMATION FOR SEQ ID NO: 2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Primer B25P variant    (B) LOCATION: anneals to nucleotides 15-33 of exon 2 of e.g.    HLA-B*4001    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:    AGGTATTTCCACACCGCCA19    (2) INFORMATION FOR SEQ ID NO: 3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Primer B25P variant    (B) LOCATION: anneals to nucleotides 15-33 of exon 2 of    HLA-B*0801    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:    AGGTATTTCGACACCGCCA19    (2) INFORMATION FOR SEQ ID NO: 4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: YES    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Primer B23P1    (B) LOCATION: anneals to nucleotides 241-261 of exon 2 of e.g.    HLA-B*4001    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:    TCTGGTTGTAGTAGCCGCGCA21    (2) INFORMATION FOR SEQ ID NO: 5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: YES    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Primer B23P2    (B) LOCATION: anneals to nucleotides 241-261 of exon 2 of e.g.    HLA-B*1301    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:    TCTGGTTGTAGTAGCGGAGCG21    (2) INFORMATION FOR SEQ ID NO: 6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: YES    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Primer B23P3    (B) LOCATION: anneals to nucleotides 219-237 of exon 2 of e.g.    HLA-B*5101    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:    TCCGCAGGTTCTCTCGGTA19    (2) INFORMATION FOR SEQ ID NO: 7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Probe 1    (B) LOCATION: anneals to nucleotides 61-78 of exon 2 of e.g.    HLA-B*4001    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:    GCTTCATCACCGTGGGCT18    (2) INFORMATION FOR SEQ ID NO: 8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Probe 2    (B) LOCATION: anneals to nucleotides 60-77 of exon 2 of e.g.    HLA-B*0801    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:    CGCTTCATCTCAGTGGGC18    (2) INFORMATION FOR SEQ ID NO: 9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Probe 3    (B) LOCATION: anneals to nucleotides 60-77 of exon 2 of e.g.    HLA-B*5101    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:    CGCTTCATTGCAGTGGGC18    (2) INFORMATION FOR SEQ ID NO: 10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Probe 4    (B) LOCATION: anneals to nucleotides 60-77 of exon 2 of e.g.    HLA-B*3501    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:    CGCTTCATCGCAGTGGGC18    (2) INFORMATION FOR SEQ ID NO: 11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Probe 5    (B) LOCATION: anneals to nucleotides 126-143 of exon 2 of e.g.    HLA-B*1501    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:    CCGAGGATGGCGCCCCGG18    (2) INFORMATION FOR SEQ ID NO: 12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Probe 6    (B) LOCATION: anneals to nucleotides 125-142 of exon 2 of e.g.    HLA-B*3501    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:    TCCGAGGACGGAGCCCCG18    (2) INFORMATION FOR SEQ ID NO: 13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Probe 7    (B) LOCATION: anneals to nucleotides 123-140 of exon 2 of e.g.    HLA-B*0801    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:    AGTCCGAGAGAGGAGCCG18    (2) INFORMATION FOR SEQ ID NO: 14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Probe 8    (B) LOCATION: anneals to nucleotides 143-161 of exon 2 of e.g.    HLA-B*5401    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:    GCGCCGTGGGTGGAGCAG18    (2) INFORMATION FOR SEQ ID NO: 15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Probe 9    (B) LOCATION: anneals to nucleotides 178-195 of exon 2 of e.g.    HLA-B*0801    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:    GGGACCGGAACACACAGA18    (2) INFORMATION FOR SEQ ID NO: 16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Probe 10    (B) LOCATION: anneals to nucleotides 179-196 of exon 2 of e.g.    HLA-B*4001    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:    GGACCGGGAGACACAGAT18    (2) INFORMATION FOR SEQ ID NO: 17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Probe 11    (B) LOCATION: anneals to nucleotides 179-196 of exon 2 of e.g.    HLA-B*5701    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:    GGACGGGGAGACACGGAA18    (2) INFORMATION FOR SEQ ID NO: 18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Probe 12    (B) LOCATION: anneals to nucleotides 189-206 of exon 2 of    HLA-B*4601    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:    ACACAGAAGTACAAGCGC18    (2) INFORMATION FOR SEQ ID NO: 19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Probe 13    (B) LOCATION: anneals to nucleotides 192-209 of exon 2 of    HLA-B*4001    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:    CAGATCTCCAAGACCAAC18    (2) INFORMATION FOR SEQ ID NO: 20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Probe 14    (B) LOCATION: anneals to nucleotides 191-208 of exon 2 of e.g.    HLA-B*0801    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:    ACAGATCTTCAAGACCAA18    (2) INFORMATION FOR SEQ ID NO: 21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Probe 15    (B) LOCATION: anneals to nucleotides 192-209 of exon 2 of e.g.    HLA-B*5401    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:    CAGATCTACAAGGCCCAG18    (2) INFORMATION FOR SEQ ID NO: 22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Probe 16    (B) LOCATION: anneals to nucleotides 191-208 of exon 2 of e.g.    HLA-B*7901    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:    ACAGATCTGCAAGACCAA18    (2) INFORMATION FOR SEQ ID NO: 23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Probe 17    (B) LOCATION: anneals to nucleotides 212-229 of exon 2 of e.g.    HLA-B*7801    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:    ACAGACTGACCGAGAGAG18    (2) INFORMATION FOR SEQ ID NO: 24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Probe 18    (B) LOCATION: anneals to nucleotides 211-228 of exon 2 of e.g.    HLA-B*4001    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:    CACAGACTTACCGAGAGA18    (2) INFORMATION FOR SEQ ID NO: 25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: Oligonucleotide Probe 19    (B) LOCATION: anneals to nucleotides 220-237 of exon 2 of e.g.    HLA-B*4001    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:    ACCGAGAGAGCCTGCGGA18    (2) INFORMATION FOR SEQ ID NO: 26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:    GTCCGAGGAAGGAGCCGC18    (2) INFORMATION FOR SEQ ID NO: 27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:    CTTCATCACCGTGGGCT17    (2) INFORMATION FOR SEQ ID NO: 28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:    GCTTCATCTCAGTGGGC17    (2) INFORMATION FOR SEQ ID NO: 29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:    GCTTCATTGCAGTGGGC17    (2) INFORMATION FOR SEQ ID NO: 30:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:    CTTCATCGCAGTGGGC16    (2) INFORMATION FOR SEQ ID NO: 31:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:    GATGGCGCCCCGG13    (2) INFORMATION FOR SEQ ID NO: 32:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:    GGACGGAGCCCCG13    (2) INFORMATION FOR SEQ ID NO: 33:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:    GAGGATGGCGCCCCGG16    (2) INFORMATION FOR SEQ ID NO: 34:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:    CGAGGACGGAGCCCCG16    (2) INFORMATION FOR SEQ ID NO: 35:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:    CCGAGGACGGAGCCCCG17    (2) INFORMATION FOR SEQ ID NO: 36:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:    TCCGAGGACGGAGCCCCGG19    (2) INFORMATION FOR SEQ ID NO: 37:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:    TCCGAGAGAGGAGCC15    (2) INFORMATION FOR SEQ ID NO: 38:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:    AGTCCGAGAGAGGAGCC17    (2) INFORMATION FOR SEQ ID NO: 39:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:    CCGTGGGTGGAGCAG15    (2) INFORMATION FOR SEQ ID NO: 40:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:    CGCCGTGGGTGGAGCAG17    (2) INFORMATION FOR SEQ ID NO: 41:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:    GGACCGGAACACACAGA17    (2) INFORMATION FOR SEQ ID NO: 42:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:    GACCGGAACACACAGA16    (2) INFORMATION FOR SEQ ID NO: 43:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:    GACCGGAACACACAG15    (2) INFORMATION FOR SEQ ID NO: 44:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:    GGACCGGAACACACAG16    (2) INFORMATION FOR SEQ ID NO: 45:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:    GACCGGGAGACACAGAT17    (2) INFORMATION FOR SEQ ID NO: 46:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:    ACGGGGAGACACGGAA16    (2) INFORMATION FOR SEQ ID NO: 47:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:    CACAGATCTTCAAGACCAAC20    (2) INFORMATION FOR SEQ ID NO: 48:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:    ACAGATCTTCAAGACCAAC19    (2) INFORMATION FOR SEQ ID NO: 49:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:    CAGATCTTCAAGACCAA17    (2) INFORMATION FOR SEQ ID NO: 50:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:    CACAGATCTGCAAGACCAA19    (2) INFORMATION FOR SEQ ID NO: 51:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:    CGAGAGAGCCTGCGG15    (2) INFORMATION FOR SEQ ID NO: 52:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:    CGAGAGAGCCTGCGGA16    (2) INFORMATION FOR SEQ ID NO: 53:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 9    (D) OTHER INFORMATION: /standard.sub.-- name= "G or C"    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 12    (D) OTHER INFORMATION: /standard.sub.-- name= "T or A"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:    GACGACACSCCWGTTCGTGA20    __________________________________________________________________________

We claim:
 1. A solid support carrying on its surface at least one probeselected from SEQ ID NO:s 7-26.
 2. The solid support according to claim1, said solid support carrying on its surface two probes, said supportbeing a membrane strip and said probes being arranged on said strip inparallel lines.
 3. A composition comprising at least one of theamplification primers selected from 5'-AGGTATTTCTACACCGCCA-3' (B25P, SEQID NO: 1), 5'-AGGTATTTCCACACCGCCA-3'(SEQ ID NO:2), and5'-AGGTATTTCGACACCGCCA-3' (SEQ ID NO:3), and optionally, at least one ofthe oligonucleotide amplification primers selected from the list5'-TCTGGTTGTAGTAGCCGCGCA-3' (B23P1, SEQ ID NO:4),5'-TCTGGTTGTAGTAGCGGAGCG-3' (B23P2, SEQ ID NO:5),5'-TCCGCAGGTTCTCTCGGTA-3' (B23P3, SEQ ID NO:6).
 4. A compositioncomprising at least one oligonucleotide probe selected from SEQ ID NO:s7-52.